Page 132 - 2020_11-Haematologica-web

N.A.G. Graça et al.
Introduction
Thrombotic thrombocytopenic purpura (TTP) is a life- threatening rare disorder triggered by a lack of activity of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). A limited number of cases are congenital, however approximately 95% of cases are of an acquired, autoimmune nature – immune TTP – in which autoantibodies targeting ADAMTS13 cause loss of enzyme activity resulting in accumulation of highly pro-thrombotic ultra-large VWF multimers.1 ADAMTS13 is a large, complex enzyme com- prising 14 domains which, from the N- to C-terminal, are: metalloprotease (M), disintegrin (D), thrombospondin- type 1-repeat 1 (TSP1), cysteine-rich (C), spacer (S), seven thrombospondin-type 1-repeats 2-8 (TSP2-8), and two CUB domains (CUB1 and CUB2)2 (Figure 1). The mecha- nisms for the loss of self-tolerance towards ADAMTS13 are not completely understood, but include specific HLA alleles, ethnicity and other genetic traits.3 Additionally, onset has also been associated with infections,4,5 drugs,6
and cases of envenomation.7,8 Most autoantibodies of patients with immune TTP are encoded by the heavy chain variable region genes VH1-699-12 and VH1-3.12 However, the immune response is polyclonal,13 usually targeting the spacer domain,14 and may target other domains as well.15-17 Antibodies targeting the MDTCS domains physically block interactions between ADAMTS13 and VWF.18 So far, isolated autoantibodies against C-terminal TSP2-8 and CUB1-2 domains have not shown a clear direct inhibitory action, at least in static assays.10 Both types can increase the clearance of ADAMTS13, which is considered the major mechanism inducing loss of ADAMTS13 activity.17,19,20
Several epitope mapping studies revealed that in the exosite-3 of the spacer domain, an epitope comprising residues R568/F592/R660/Y661/Y665 (RFRYY) is com- monly targeted in nearly 95% of patients with immune TTP (Online Supplementary Table S1).10,15-17,21-24 Studies of spacer exosite-3 alanine scans have shown that alanine modifications lead to reductions in exosite-3 spacer autoantibody binding,22,23 but also result in reductions of activity.23,25 Another study revealed that highly conserva-
Figure 1. Overview of all ADAMTS13 spacer exosite-3 mutant variants created for this study. Full-length and MDTCS ADAMTS13 variants used in this study are shown. For ease and direct comparison with the original exosite-3 epitope (RFRYY), the mutations inserted are shown with the one-letter amino acid code in different colors and underlined. A total of 40 full-length variants were used, comprising conservative (Y↔F), semi-conservative (F/Y→L), non-conservative (F/Y→N), classic alanine (F/Y/R→A) and alanine/lysine hybrid mutations (F/Y→A + R→K). The residue substitutions were chosen based on the physicochemical characteristics of the amino acid side-chains (see Online Supplementary Methods for a more detailed description of the rationale for the different choices of amino acids). The classic gain-of-function (KYKFF) variant was also included in this study. In addition, two MDTCS variants were included (lower panel).
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haematologica | 2020; 105(11)